Non-invasive measurement of blood analytes using photodynamics

ABSTRACT

The determination of blood glucose in an individual is carried out by projecting illuminating light into an eye of the individual to illuminate the retina with the light having wavelengths that are absorbed by rhodopsin and with the intensity of the light varying in a prescribed temporal manner. The light reflected from the retina is detected to provide a signal corresponding to the intensity of the detected light, and the detected light signal is analyzed to determine the changes in form from that of the illuminating light. For a biased sinusoidal illumination, these changes can be expressed in terms of harmonic content of the detected light. The changes in form of the detected light are related to the ability of rhodopsin to absorb light and regenerate, which in turn is related to the concentration of blood glucose, allowing a determination of the relative concentration of blood glucose. Other photoreactive analytes can similarly be determined by projecting time varying illuminating light into the eye, detecting the light reflected from the retina, and analyzing the detected light signal to determine changes in form of the signal due to changes in absorptivity of a photoreactive analyte.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of prior application Ser. No.10/012,902, filed Oct. 22, 2001, which claimed priority from provisionalapplication No. 60/318,850, filed Sep. 13, 2001, which are incorporatedherein by reference.

FIELD OF THE INVENTION

This invention pertains to the field of non-invasive in vivo measurementof blood analytes.

BACKGROUND OF THE INVENTION

The measurement of blood glucose by diabetic patients has traditionallyrequired the drawing of a blood sample for in vitro analysis. The bloodsampling is usually done by the patient himself as a finger puncture, orin the case of a child, by an adult. The need to draw blood for analysisis undesirable for a number of reasons, including discomfort to thepatient, resulting in many patients not testing their blood asfrequently as recommended, the high cost of glucose testing supplies,and the risk of infection with repeated skin punctures.

Many of the estimated three million Type 1 juvenile) diabetics in theUnited States are asked to test their blood glucose six times or moreper day in order to adjust their insulin doses for tighter control oftheir blood glucose. As a result of the discomfort, many of thesepatients do not test as often as is recommended by their physician, withthe consequence of poor blood glucose control. This poor control hasbeen shown to result in increased complications from this disease. Amongthese complications are blindness, heart disease, kidney disease,ischemic limb disease, and stroke. In addition, there is recent evidencethat Type 2 (adult-onset) diabetics (numbering over 10 million in theUnited States) may reduce the incidence of diabetes-relatedcomplications by more tightly controlling their blood glucose.Accordingly, these patients may be asked to test their blood glucose asoften as the Type 1 diabetic patients.

It would thus be desirable to obtain fast and reliable measurements ofthe blood glucose concentration through simple, non-invasive testing.Prior efforts have been unsuccessful in the quest for a sufficientlyaccurate, non-invasive blood glucose measurement. These attempts haveinvolved the passage of light waves through solid tissues such as thefingertip and the ear lobe and subsequent measurement of the absorptionspectra. These efforts have been largely unsuccessful primarily due tothe variability of absorption and scatter of the electromagnetic energyin the tissues. Other groups have attempted blood glucose measurement inbody fluids such as the anterior chamber, tears, and interstitialfluids. To date, these efforts have not been successful for a variety ofreasons.

SUMMARY OF THE INVENTION

The present invention combines the accuracy of in vitro laboratorytesting of analytes such as blood glucose with the advantages of arapidly-repeatable non-invasive technology. The invention utilizes ahand-held instrument that allows non-invasive determination of glucoseby measurement of the regeneration rate of rhodopsin, the retinal visualpigment, following a light stimulus. The rate of regeneration ofrhodopsin is dependent upon the blood glucose concentration, and bymeasuring the regeneration rate of rhodopsin, blood glucose can beaccurately determined. This invention exposes the retina to light ofselected wavelengths in selected distributions and subsequently analyzesthe reflection from the exposed region.

The rods and cones of the retina are arranged in specific locations inthe back of the eye, an anatomical arrangement used in the presentinvention. The cones, which provide central and color vision, arelocated with their greatest density in the area of the fovea centralisin the retina. The fovea covers a circular area with a diameter of about1.5 mm, with a subtended angle of about 3 degrees. The rods are found inthe more peripheral portions of the retina and contribute to dim vision.

The light source in the invention that is used to generate theilluminating light is directed on the cones by having the subject lookat the light. This naturally provides for the incident light strikingthe area of the retina where the cones (with their particular rhodopsin)are located. The incoming light preferably subtends an angle muchgreater than the angle required to include the area of the foveacentralis, so that the entire reflected signal includes the area of highcone density.

The invention uses light that varies in a selected temporal manner, suchas a periodically applied stimulus of light (for example, a sinusoidalpattern), and then analyzes the reflected light from the retina todetermine the distortion of the detected light relative to theilluminating light. The excitation format chosen allows removal of thelight signal due to passive reflection. For example, the primaryfrequency of an applied sinusoidal stimulus can be filtered out of thelight received back from the eye, leaving higher order harmonics of thefundamental as the input into the analysis system (for example, a neuralnetwork). Measurement of unknown blood glucose concentration isaccomplished by development of a relationship between these input dataand corresponding clinically determined blood glucose concentrationvalues.

Similarly, this technique can be utilized in the analysis ofphotoreactive analytes such as bilirubin. Bilirubin is a molecule thatis elevated in a significant number of infants, causing newbornjaundice. It would be desirable to non-invasively measure bilirubin, asthis is currently done with invasive blood testing. This moleculeabsorbs light at 470 nm and exhibits a similar photo-decomposition torhodopsin, but without regeneration. In a manner similar to thatdescribed above for rhodopsin measurement, bilirubin may be measuredutilizing a time-varying light signal and analyzing the correspondingreflected light signals for non-passive responses due tophoto-decomposition. More generally, an analysis—model-based orstatistical—of descriptors (amplitude, polarization, transient orharmonic content) of incident and detected light can be carried out todetermine a variation in the detected signal resulting fromlight-induced changes in the physical or chemical interaction of aphotoreactive analyte with the illuminating light.

In accordance with the invention, a hand-held or stationary instrumentthat measures the resulting data in the reflected light from aperiodically applied light stimulus (for example, a sinusoid) may beutilized for the determination of blood glucose values. There may bepatient-to-patient variability and each device may be calibrated foreach patient on a regular interval. This may be necessary as thechanging state of each patient's diabetes affects the outer segmentmetabolism and thus influences the regeneration rates of rhodopsin. Theintermittent calibration of the device is useful in patient care as itfacilitates the diabetic patient returning to the health-care providerfor follow-up of their disease. The device may be equipped with a methodof limiting the number of tests, so that follow-up will be required toreactivate the device.

In the present invention, the reflected light data may be sent to acentral computer by a communications link in either a wireless or wiredmanner for central processing of the data. The result may then be sentback to the device for display or be retained to provide a historicalrecord of the individual's blood glucose levels.

Further objects, features, and advantages of the invention will beapparent from the following detailed description when taken inconjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

In the drawings:

FIG. 1 is a schematic diagram of an apparatus for measurement of theconcentration of blood glucose in accordance with the invention.

FIG. 2. is a schematic diagram of the preferred embodiment of theillumination and optical system of the apparatus of FIG. 1.

FIG. 3 is an illustrative diagram of the amplitude of the input signalfrom the illumination source.

FIG. 4 is a graph illustrating the results of a mathematical simulationof the time response of the light-related biochemistry reflected fromthe fovea centralis.

FIG. 5 is a graph of the harmonic content of the reflected light fromthe fovea centralis.

FIG. 6 is a schematic side view of a hand-held measurement system thatmay be utilized in accordance with the invention.

FIG. 7 is a schematic diagram of another embodiment of an illuminationand optical system that may be utilized in the invention.

FIG. 8 is a schematic diagram of an embodiment of an illumination andoptical system utilizing a polarizing cube beamsplitter for reducing theeffect of unscattered reflections.

FIG. 9 is a schematic diagram of an embodiment of an illumination andoptical system utilizing a reflecting mirror with an aperture forreducing the effect of unscattered reflections.

DETAILED DESCRIPTION OF THE INVENTION

Rhodopsin is the visual pigment contained in the rods and cones of theretina. As this pigment absorbs light, it breaks down into intermediatemolecular forms and initiates a signal that proceeds down a tract ofnerve tissue to the brain, allowing for the sensation of sight. Theouter segments of the rods and cones contain large amounts of rhodopsin,stacked in layers lying perpendicular to the light incoming through thepupil. There are two types of rhodopsin, with a slight differencebetween the rhodopsin in the rods (that allow for dim vision) and therhodopsin in the cones (that allow for central and color vision). Rodrhodopsin absorbs light energy in a broad band centered at 500 nm,whereas there are three different cone rhodopsins having broadoverlapping absorption bands peaking at 430, 550, and 585 nm.

Rhodopsin consists of 11-cis-retinal and the protein opsin, which istightly bound in either the outer segment of the cones or rods.11-cis-retinal is the photoreactive portion of rhodopsin, which isconverted to all-trans-retinal when a photon of light in the activeabsorption band strikes the molecule. This process goes through asequence of chemical reactions as 11-cis-retinal isomerizes toall-trans-retinal. During this series of chemical steps, the nervefiber, which is attached to that particular rod or cone, undergoes astimulus that is perceived in the brain as a visual signal.

Following the breakdown of 11-cis-retinal to all-trans-retinal, the11-cis-retinal is regenerated by a series of steps that result in11-cis-retinal being recombined with opsin protein in the cell or diskmembrane. A critical step in this regeneration pathway is the reductionof all-trans-retinal to all-trans-retinol, which requires NADPH as thedirect reduction energy source. In a series of experiments, Futterman etal have proven that glucose, via the pentose phosphate shunt (PPS),provides virtually all of the energy required to generate the NADPHneeded for this critical reaction. S. Futterman, et al., “Metabolism ofGlucose and Reduction of Retinaldehyde Retinal Receptors,” J.Neurochemistry, 1970, 17, pp. 149-156. Without glucose or its immediatemetabolites, no NADPH is formed and rhodopsin cannot regenerate.

There is strong evidence that glucose is a very important energysubstrate for the integrity and function of the retinal outer segments.It has been known since the 1960s that glucose and glycolysis (themetabolism of glucose) are important in maintaining the structure andfunction of the retinal outer segments. More recently, it has beendiscovered that one of the major proteins contained in the retinal outersegments is glyceraldehyde-3-phosphate dehydrogenase, an importantenzyme in glucose metabolism. This points to the importance of glucoseas the energy source for the metabolism in the retinal outer segments,which has as its primary function the maintenance of high concentrationsof rhodopsin.

In addition, Ostroy, et al. have proven that the extracellular glucoseconcentration has a major effect on rhodopsin regeneration. S. E.Ostroy, et al., “Extracellular Glucose Dependence of RhodopsinRegeneration in the Excised Mouse Eye,” Exp. Eye Research, 1992, 55, pp.419-423. Since glucose is the primary energy driver for rhodopsinregeneration, the present invention utilizes this principle to measureextracellular glucose concentrations.

Furthermore, recent laboratory work by Ostroy et al has shown that theretinal outer segments become acidic with chronic elevated blood glucoseconcentrations. S. E. Ostroy, et al., “Decreased Rhodopsin Regenerationin Diabetic Mouse Eyes,” Invest. Ophth. and Visual Science, 1994, 35,pp. 3905-3909; S. E. Ostroy, et al., “Altered Rhodopsin Regeneration inDiabetic Mice Caused by Acid Conditions Within Rod Receptors,” CurrentEye Research, 1998, 17, pp. 979-985. Work in McConnell's laboratory hascharacterized the retinal outer segments with these diabetic pH changes.It has been noted that with increasing acidity of the retinal outersegments, there exist pronounced changes in the light scattering by thecells. These experiments reveal that as blood glucose increasesintracellular pH decreases. These changes affect the absorption spectraand the light scattering properties of these cells and are directlydetermined by intracellular glucose concentration. This scatteringeffect is measured with the present invention and adds an additionalvariable in the reflection of light, driven by the glucoseconcentration, providing for even further accuracy with this invention.

The following is an analysis of the photodynamic reactions associatedwith the present invention:

Define:

-   -   R₀=molecules/unit volume of rhodopsin    -   R₁=molecules/unit volume of all-trans-retinal isomer    -   G=molecules/unit volume of cytosol (intracellular) glucose    -   G₀=molecules/unit volume of extracellular glucose    -   L=photons/cm² sec incident on the fovea

Recognizing that there are other photodynamic reactions involved, asimple and conceptually accurate representation of the rhodopsin cycleis given by the following equations:dR ₀ /dt=−k ₁ R ₀ L+k ₂R₁ G  Equation 1dR ₁ /dt=k ₁ R ₀ L−k ₂ R ₁ G  Equation 2dG/dt=k ₃(G ₀ −G)−k ₂ R ₁ G  Equation 3

An auxiliary equation links the observed reflectance, R_(F), of thefoveal region to R₀. Let R_(F min) be the reflectance under the fillybleached conditions and R_(F max) be the reflections when unbleached.Then the foveal reflectance is approximately:R _(F) =R _(F min)+(R _(F max) −R _(F min))e ^(−k) ⁴ ^((R) ^(dark) ^(−R)⁰ ⁾  Equation 4

-   -   where:    -   R_(F max) reflection at near dark conditions    -   R_(F min) reflection at fully bleached conditions    -   R_(dark) maximum value of R₀

The R_(F min) value is reflectance of the pigment epithelium, which is adark layer of tissue directly underneath the rods and cones. R_(F max)is determined by the optical characteristics of the absorption process.R_(dark) and k₄ can be determined from historical measurement data. Thepoint is that foveal reflectance varies in a predictable way with R₀ andhence with L and G. This variation is exploited in the present inventionto remove noise during analysis of reflected light; if the fovea isexposed to sinusoidally varying amplitude of light, then, because of theabove noted variation of reflectance, the reflected light will containharmonics of the frequency of variation of the incident light for thefoveal reflections which vary with bleaching. All the passivelyreflected light will have amplitude varying at the frequency of theincident light.

Since the harmonics of the incident light frequency contain the neededinformation about R₀, the fundamental frequency can be removed by datafiltering techniques. This restricts analyzed data to light reflectedfrom the active foveal cells, greatly improving signal to noise ratios.

The data gathering and analysis process illuminates the posterior retinawith light capable of bleaching rhodopsin and varies the lightamplitude, preferably sinusoidally, at an appropriate rate or frequency(or multiple rates). Light reflected in part from the anterior retina isthen examined for intensity/amplitude at 2,3,4, etc. times the frequencyof variation of the incident light. The estimated amplitudes of theharmonics are closely related to the bleaching process, which is knownto depend upon cellular glucose concentrations as discussed above.Harmonic amplitudes can be related to measured glucose concentrationswith a number of regression techniques or by the use of artificialneural network methods.

A simple example of this idea is the following:

Assume that foveal reflectance R_(F) is linearly related to incidentlight amplitude L: L=A sin 2πft, and R_(F)=BL=AB sin 2πft

Then, R_(F)L=A²B sin²2πft=A²B(1/2−1/2 cos 4πft)

The reflected light is thus seen to be a constant amplitude componentand a component varying with twice the incident frequency.

With reference to the drawings, FIG. 1 illustrates a glucose analysisapparatus in accordance with the invention in conjunction with the eyeof a patient, with the eye shown illustratively at 10 in FIG. 1. Theglucose analysis apparatus includes an illumination and optics system 15comprised of a light source and lens system for projecting illuminatinglight onto the fundus, directly through the pupil, and for receiving thelight reflected from the fundus passed out through the pupil. The lensespreferably include a final lens which can be positioned close to thecornea of the eye, providing a 5 to 30 degree conical view of the retinato be illuminated and the light reflected back to the illumination andoptics system 15.

The illuminating light from the illumination and optics system 15includes a time varying (modulated) light amplitude (preferablysinusoidal) added to (constant) amplitude of at least half of thesinusoidal peak to peak value, as illustrated in FIG. 3. The wavelengthrange of the illuminating light preferably matches the active range ofthe rhodopsin molecules illuminated. Several frequencies of modulationof the illumination light from the illumination and optics system 15,e.g., three frequencies of input light, are preferably utilized inserially applied tests to provide multiple sets of information tocharacterize the reflectance from the retina. Illumination light may beprovided by various light sources, for example, a xenon light, a lightemitting diode (LED), or a halogen light source. LED illumination ispreferred because of the ease of varying the intensity of the light fromthe LED by varying the input power to the LED. Alternatively, steadystate sources may be used with light modulators to provide theappropriate time varying illumination. The patient being tested may bedirected to look directly at the light source, and by centering thefield of view on the incoming light, the appropriate area of the fundus(fovea centralis) will be illuminated. Since the area of interest issmall compared to the area that is illuminated, it is generally notcritical that the illuminating light strikes the fundus at anyparticularly exact area of the retina. Furthermore, since the area ofinterest is in the approximate center of the area illuminated, thecorrect area is easily illuminated. Although the invention may becarried out with a dilated eye pupil, it is an advantage of the presentinvention that the testing can be carried out without requiring dilationof the pupil for speed of measurement and patient convenience.

The illuminating light reflected from the fundus of the eye 10 passesout through the pupil opening of the eye to the illumination and opticssystem 15, entering a (preferably) single element photodetector 16, asillustrated in FIG. 1. Optical data (e.g., in the form of an analogelectrical signal or a digitized signal) from the single elementphotodetector 16 is provided to the optical data analysis system 17,where the information on the reflected light is processed with, e.g., aphase-locked loop at 2, 3, and 4 times the light input modulationfrequency. This provides analysis of the higher order harmonics, whichwill be described in more detail below.

The data in the reflected primary frequency of light (containing noiseincluding optical system and eye reflections) is preferably not used.Only harmonics of the primary frequency are preferably utilized as datainput to a processor that carries out a calculation of the blood glucoseconcentration. There are various methods to eliminate the primaryfrequency of light including passive filtering, phase lock loop, andmany digital processing techniques. Alternatively, a signal analysissuch as a fast Fourier transform can be performed and subsequently onlythe higher harmonics may be used as data input. An additional variable,associated with the light scattering effect of chronically high glucoseconcentrations on the outer segments of the retina, affects thereflected light data and can be accounted for in the processing of thedata.

The optical reflectance measurements may then be correlated with bloodglucose concentration measurements. Fast Fourier Transforms (FFT) of theharmonic content data along with patient calibration data from a datastorage 18 may, for example, be utilized in a neural network simulationcarried out by computer. Exemplary neural network and FFT analysis toolsthat may be used in one embodiment of the invention are contained in theMATLAB™ language and in the Neural Network Toolbox of MATLAB™ version12.1. The neural network iteratively generates weights and biases whichoptimally represent, for the network structure used, the relationshipbetween computed parameters of the detected light signal and bloodglucose values determined by the usual methods. The desired relationshipmay be amenable, alternatively, to development as a look-up table,regression model, or other algorithm carried out in the optical dataanalysis system 17, e.g., a special purpose computer or an appropriatelyprogrammed personal computer, work station, etc.

The relationship between the optical measurements made using theapparatus of the invention and measurement made on blood samples takenfrom the individual patient may change over a period of time. Thepatient calibration data in the data storage 18 may be combined with analgorithm carried out in the optical data analysis system 17 to predictthe specific patient's blood glucose concentration, and the calibrationdata may be periodically updated. The health-care provider may performperiodic calibration of the apparatus at certain intervals, preferablyevery three months.

The results of the calculated blood glucose concentration from theoptical data analysis system 17 are provided to an output system 19 forstorage, display or communication. A readout of the glucoseconcentration history from a data history storage 20 may be obtained bythe health care provider at convenient intervals. The blood glucoseconcentration may be directed from the output system 19 to a display 21to provide for patient observation. This display 21 will be preferablyby an LCD screen located on the device as depicted as 21 in FIG. 6.

FIG. 2 shows a schematic diagram of a preferred embodiment of theoptical system 15. Illuminating sinusoidal light is generated by an LED13 and coupled to one leg of a dual branch fiberoptic light guide 12. Anexample of an LED that may be used is a Gilway E903 green LED, and thedual branch fiberoptic light guide may be the Edmund Industrial Opticslight guide #L54-200. The illuminating light has wavelength contentpreferably consistent with the wavelengths known to activate rhodopsinin areas of the fovea illuminated by the incoming light. These preferredwavelengths are in the range of 500 nm to 580 nm. This illuminatinglight is amplitude modulated to a sinusoidal shape as depicted in FIG.3. Illuminating light from the LED 13 passes through the dual branchfiberoptic light guide 12 and is delivered to a lens 11, which thenpasses the light through the pupil of the eye 10 of the patient and ontothe retina. The retina, including the fovea centralis, is flooded withilluminating light. The illuminating light is then reflected from theretina, passes out through the pupil, and enters the lens 11 where thelight re-enters the light guide 12. Since the light that enters the dualbranch fiberoptic light guide 12 near the lens 11 will be split at they-portion of the dual branch fiberoptic light guide 12, approximately50% of the reflected light will be presented to the photodetector 14.The photodetector 14 of FIG. 2 corresponds to the single elementphotodetector 16 of FIG. 1.

An alternative to the above-described embodiment of the optical systemincludes a conventional lens system which is used to direct theillumination light to the pupil and the returned reflected light fromthe retina may be transported on this conventional lens system (a commonpath). The reflected light may then be directed to the photodetector bythe use of a beamsplitter.

Another embodiment of the optical system 15 is shown in FIG. 7. A hybriddevice 28 is utilized that contains both an LED 13 and a photodetector14 in a common container. The LED 13 and the photodetector 14 areoptically isolated by a barrier 29. The lens 11 is positioned such thatlight from the LED 13 illuminates an out of focus area on the retina,and that area is reflected onto the photodetector 14. The operation ofthis optical system is the same as described for FIG. 2 above.

FIG. 3 is a depiction of the input modulation signal for theillumination source. The time varying signal is preferably a sinusoidwith a constant bias sufficient to prevent the waveform from reachingzero signal. While a non-sinusoidal signal (e.g., a square wave, etc.)or a signal reaching zero amplitude could be used, the biased puresinusoid (a sine wave and a constant component) is preferred forsimplicity of data analysis. The wavelength of the illuminating light ispreferably in the range of 500 nm to 600 nm, e.g., 550 nm, for analysisof glucose, although other wavelength ranges may be utilized asappropriate. The modulation frequencies of these input signals arepreferably in the range of 0.1 to 200 cycles per second (Hz) andmultiple frequencies may be utilized during the test period. Theilluminating light is preferably applied at different modulationfrequencies during the test period, for example, three sequential testsusing 1, 3, and 10 Hz, a total test period of approximately 15 secondsand with 10 cycles test duration at each frequency.

FIG. 4 shows the results of a computer implemented mathematicalsimulation of the individual responses of particular molecules in theretina. Time is displayed on the abscissa (in seconds), while theordinate depicts the relative concentrations of the particular analytes,shown as relative absorbance of light energy. The upper curve models theresponse of the rhodopsin and 11-cis-retinal (the photoreactive portion)as the rhodopsin is bleached by the sinusoidal illuminating light fromthe LED. The lower curve simulates the concentrations of opsin and11-trans-retinal, which regenerates into the 11-cis-retinal. The middlecurve reveals the consumption of the cytosol glucose, which is the soleenergy source for the regeneration of the 11-cis-retinal. The rates ofbleaching and regeneration shown in the upper and lower curves aredriven by the amount of glucose available in the cells to supportregeneration.

FIG. 5 shows the results of a Fast Fourier Transform (FFT) carried outon an example of data corresponding to light reflected from the retinaat one selected excitation frequency. The rhodopsin content and thebleaching of rhodopsin can be measured by analyzing the reflected lightfrom the retina. Since the amount of reflected light is a function ofthe amount of bleaching, and the amount of bleaching is a function ofthe intensity of incoming light, the reflection is a non-constantresponse and will contain harmonic content. The primary reflectedresponse is at the modulation frequency of the LED light output. Thisprimary frequency is preferably filtered out, e.g., with a digital highpass filter that is set to filter all frequency content less than twicethe illuminating frequency, because it contains noise generated byreflections from the optical system and the layers of the eye. Theremaining higher order harmonics contain noise-free information and areused as input data to a computer processing system, e.g., implementing aneural network, along with patient calibration data. By training theneural network with data from a large number of patients, theappropriate relationships and weighting factors are determined. Thesevalues are used in development of an algorithm that accurately predictsblood glucose concentrations from the available information.

An illustration of a hand-held device embodying the invention is shownin FIG. 6. This unit contains the elements depicted in FIG. 1 and FIG.2. The display 21 provides the glucose concentration information to thepatient, preferably utilizing an LCD screen. An electrical connector 23can be utilized by the patient or healthcare provider to cable connectto a host system that allows for reading out of the data history fromthe storage 20 (see FIG. 1) and updating of the patient calibration datain the storage 18 (FIG. 1). A button 24 is provided to activate the unitfor data acquisition, in a manner similar to taking a photograph with astandard camera. If desired, a disposable plastic cover can be used tocover the lens 11 to minimize the spread of infectious diseases. Thehand-held unit is preferably self-contained and contains batteries andmemory.

The analysis of the reflected signal may take place at a location remotefrom the clinical setting by using a wired or wireless internet link (ordedicated communication link) to transfer data from the photodetector toa central computer at a remote location (e.g., anywhere in the worldlinked by the internet) where the optical data analysis system 17 (seeFIG. 1) is implemented. The output data from the output system 19 may betransferred back through an access link to the display of results 21 orto another location if desired.

The illumination and optics systems of FIGS. 2 and 7 provide a means forprojecting illuminating light into the eye with an intensity varyingperiodically at a selected frequency and means for detecting the lightreflected from the retina and particularly the fovea centralis toprovide a signal corresponding to the intensity of the detected light.Any other elements which similarly project light into the eye and detectthe reflected light may be utilized. Examples of such alternative meansare illustrated in FIGS. 8 and 9, although it is understood that theseare exemplary only of such structures. The illumination and opticssystems of FIGS. 8 and 9 are arranged to help reduce the intensity ofthe light reflected from structures of the eye other than the retina,and particularly to reduce the effect of light reflected from thesurface of the cornea. In the illumination and optics system of FIG. 8,the light projected from a source 31, such as an LED, expands in a beam32 which is received by a lens 33 which directs the beam to a polarizingbeamsplitter 35. The polarized beam 36 that exits the beamsplitter ispassed through a lens 37 and an eyepiece lens 38 to the eye 10 where thelens of the eye focuses the beam onto the fovea 39 of the retina. Thelight reflected from the retina and particularly the fovea 39 (and fromother eye structures such as the cornea 40) is directed back through theeyepiece 38 and the lens 37 to the polarizing beamsplitter 35. Thepolarized light reflected from the surface 40 of the cornea passesthrough the beamsplitter 35 and is lost, while the scattered(non-polarized) light resulting from reflection from the retina andparticularly the fovea 39 is reflected by the beamsplitter 35 into abeam 42 which is focused by a lens 43 onto a photodetector 44 thatprovides an output signal on a line 45 corresponding to the (timevarying) intensity of the detected light. This signal may then beanalyzed to determine the magnitude of a harmonic or harmonics of thefrequency of variation of the illuminating light.

In the illumination and optics system of FIG. 9, the light from a source48 (e.g., an LED) is projected in a beam 49 to a lens 50 which providesa collimated beam 51 to a mirror 53 which has a central aperture 54therein. The aperture 54 permits a central portion 55 of the beam 51 topass therethrough and be lost. The rest of the beam 51 is reflected fromthe surface 56 of the mirror 53 into a beam 57 which is received by afocusing system composed of a first lens 58 and a second lens 60 toprovide an output beam 61 that passes through the cornea 40 of the eye10 and is focused by the lens of the eye onto the retina of the eye andparticularly the fovea 39. The light reflected from the retina andparticularly the fovea 39 passes back through the lens of the eye andthe cornea 40 and into the optical system composed of the lenses 58 and60, which forms the light reflected from the fovea 39 into a beam 62 inthe center of the reflected beam. The beam of light 62 reflected fromthe fovea is narrow enough to substantially pass through the aperture 54to a lens 64 which focuses the beam onto a photodetector 65 thatprovides an output signal on a line 66 corresponding to the intensity ofthe detected light. The aperture 54 will appear as a dark spot in thefield of view of the individual being tested, and the light reflectedfrom the fovea will be naturally aligned with the aperture 54 by havingthe individual focus on the dark spot in the field of view. In theillumination and optics systems of FIG. 9, the light that reaches thedetector 65 is thus primarily the light reflected from the fovea 39 inthe relatively narrow beam portion 62, whereas the light reflected fromother structures in the eye, such as the surface of the cornea 40, iscontained in a broader reflected beam that reaches the surface of themirror 56 and is reflected thereby rather than being passed through theaperture 54, again improving the signal to noise ratio of the lightreaching the detector 65.

It is understood that the invention is not confined to the particularembodiments set forth herein for illustration, but embraces all suchforms thereof as come within the scope of the following claims.

1-28. (canceled)
 29. A method for use in the determination of the bloodglucose concentration in an individual, comprising: (a) Non-invasivelymeasuring the rate of a biochemical process of the body; (b) Determiningthe blood glucose concentration from the measured rate.
 30. The methodaccording to claim 29 wherein the biochemical process comprises aphotodynamic process.
 31. The method according to claim 29 wherein thebiochemical process comprises the reduction of all-trans-retinal toall-trans-retinol.
 32. The method according to claim 29 wherein at leasta portion of the biochemical process occurs in the eye.
 33. The methodaccording to claim 32 wherein the non-invasively measuring step occursin the eye without dilation of the pupil.
 34. The method according toclaim 29 wherein the biochemical process comprises the production ofrhodopsin.
 35. The method according to claim 29 wherein the rhodopsin iscone rhodopsin.
 36. The method according to claim 29 wherein therhodopsin is rod rhodopsin.
 37. The method according to claim 29 whereinthe non-invasively measuring step comprises measuring a rate of changein the reflectance of a surface in the eye.
 38. The method according toclaim 37 wherein the surface of the eye comprises the retina.
 39. Themethod according to claim 38 wherein the surface in the eye comprisesmostly cones.
 40. The method according to claim 38 wherein the surfacein the eye comprises rods and cones.
 41. A method of determining theblood glucose concentration in an individual, comprising: (a)non-invasively measuring a formation rate of a substance in theindividual; and (b) determining the blood glucose concentration in theindividual from the measured formation rate of the substance.
 42. Themethod according to claim 41 wherein the non-invasively measuring stepis performed in the eye.
 43. The method according to claim 41 whereinnon-invasively measuring comprises measuring the rate of change of thereflectance of a portion of the eye.
 44. The method according to claim41 wherein non-invasively measuring comprises illuminating the eye. 45.The method according to claim 41 wherein illuminating the eye comprisesilluminating the retina.
 46. The method according to claim 45 whereinilluminating the eye comprises illuminating the eye with a wavelength oflight absorbed by rod rhodopsin.
 47. The method according to claim 45wherein illuminating the eye comprises illuminating the eye with awavelength of light absorbed by cone rhodopsin.
 48. The method accordingto claim 41 wherein measuring comprises initiating a photodynamicprocess in the eye.
 49. The method according to claim 41 wherein thesubstance comprises cone rhodopsin.
 50. The method according to claim 41wherein the substance comprises rod rhodopsin.
 51. A method fordetermining the blood glucose concentration of an individual,comprising: (a) measuring a formation rate of visual pigment in an eyeof the individual; and (b) determining the blood glucose concentrationof the individual from the measured formation rate of visual pigment.52. The method according to claim 51 wherein measuring comprisesnon-invasively measuring.
 53. The method according to claim 51 whereinmeasuring a formation rate comprises measuring the rate of change of thereflectance of a portion of the eye.
 54. The method according to claim51 wherein the measuring step comprises illuminating the retina.
 55. Themethod according to claim 54 wherein the measuring step comprisesilluminating the retina using an illumination source outside of the eye.56. The method according to claim 51 wherein the measuring stepcomprises illuminating the fovea.
 57. The method according to claim 54wherein illuminating the eye comprises illuminating the eye with awavelength of light absorbed by rod rhodopsin.
 58. The method accordingto claim 54 wherein illuminating the eye comprises illuminating the eyewith a wavelength of light absorbed by cone rhodopsin.
 59. The methodaccording to claim 54 or 56 wherein illuminating the eye initiates aphotodynamic process in the eye.
 60. The method according to claim 59wherein the photodynamic process is a regenerative process.
 61. Themethod according to claim 59 wherein the photodynamic process is adepletive process.
 62. The method according to claim 60 wherein theproduct of the regenerative process is visual pigment.
 63. The methodaccording to claim 61 wherein the product of the regenerative process isvisual pigment.
 64. The method according to claim 54 wherein themeasuring step comprises illuminating the eye with a periodicallyapplied stimulus of light.
 65. The method according to claim 54 whereinthe measuring step comprises illuminating the eye without dilating thepupil.
 66. A method for determining the blood glucose concentration ofan individual, comprising: (a) Initiating a photodynamic process in theeye of a person; (b) Measuring the glucose use rate of the photodynamicprocess; and (c) Determining the blood glucose concentration of theperson from the measured glucose use rate.
 67. The method according toclaim 66 wherein the initiating step is performed without dilating thepupil.
 68. The method according to claim 66 the initiating step furthercomprising illuminating the eye with light.
 69. The method according toclaim 68 the initiating step further comprising illuminating the eyewith light having a wavelength known to activate rhodopsin.
 70. Themethod according to claim 69 the initiating step further comprisingilluminating the eye with light having a wavelength known to activaterhodopsin in areas of the fovea.
 71. The method according to claim 66wherein the biochemical process comprises the reduction ofall-trans-retinal to all-trans-retinol.
 72. The method according toclaim 66 wherein the photodynamic process causes a change in thereflectance of a portion of the eye.
 73. The method according to claim72 wherein the portion of the eye comprises the retina.
 74. The methodaccording to claim 73 wherein the portion of the eye comprises thefovea.
 75. The method according to claim 69 wherein the portion of theeye comprises a portion of the fovea.
 76. The method according to claim72 wherein the portion of the eye comprises mostly cones.
 77. The methodaccording to claim 72 wherein the portion of the eye comprises both rodsand cones.
 78. The method according to claim 66 wherein the photodynamicprocess comprises the generation of NADPH.
 79. An apparatus for glucosemeasurements, comprising: (a) An illuminating optics system adapted toprovide illuminating light into the eye at a wavelength selected toinitiate a photodynamic process in the eye; (b) An optical detectorconfigured to receive illuminating light reflected from the eye andoutput optical data relating to the photodynamic process; and (c) Anoptical data analysis system configured to process the optical data tocalculate the blood glucose level.
 80. The apparatus according to claim79 wherein the illuminating optics system provides illuminating lightinto the eye at a wavelength range matching the active range ofrhodopsin molecules.
 81. The apparatus according to claim 79 wherein theilluminating optics system provides a 5 to 30 degree conical view of theretina to be illuminated.
 82. The apparatus according to claim 79wherein the illuminating optics system provides modulated illuminatinglight.
 83. The apparatus according to claim 79 wherein the illuminatingoptics system provides illuminating light utilizing serially appliedtests.
 84. The apparatus according to claim 79 the illuminating opticssystem further adapted to provide illuminating light into the eye tocharacterize the reflectance from the retina.
 85. The apparatusaccording to claim 79 wherein the optical detector is a single elementphotodetector.
 86. The apparatus according to claim 79 wherein theoptical data analysis system calculates the blood glucose level using alook up table.
 87. The apparatus according to claim 79 wherein theoptical data analysis system calculates the blood glucose level using analgorithm.
 88. The apparatus according to claim 79 wherein the opticaldata analysis system calculates the blood glucose level using aregression model.
 89. The apparatus according to claim 79 furthercomprising: (a) data storage comprising patient calibration data. 90.The apparatus according to claim 89 wherein the patient calibration datais combined with an algorithm carried out in the optical data analysissystem to calculate the blood glucose level.
 91. The apparatus accordingto claim 89 wherein the data storage is further adapted to receiveupdated patient calibration data.
 92. The apparatus according to claim79 wherein the optical data analysis system is configured to provide anoutput for storage, display or communication.
 93. The apparatusaccording to claim 92 wherein the output comprises a readout of glucoseconcentration history.
 94. The apparatus according to claim 79 whereinthe apparatus is configured to be a hand-held device.
 95. The apparatusaccording to claim 79 wherein the optical data is processed at a remotelocation.
 96. The apparatus according to claim 95 wherein the opticaldata is sent wirelessly to be processed at a remote location.
 97. Theapparatus according to claim 95 wherein the optical data is sent via anaccess link to be processed at a remote location.
 98. The apparatusaccording to claim 79 wherein the illuminating optics system is arrangedto help reduce the intensity of the light reflected from structures ofthe eye other than the retina.
 99. The apparatus according to claim 79wherein the illuminating optics system is adapted to provide polarizedilluminating light into the eye.
 100. The apparatus according to claim79 wherein the apparatus is equipped with a method requiringreactivation of the apparatus after a limited number of uses.
 101. Amethod of determining the blood glucose concentration in an individual,comprising: (a) non-invasively measuring a rate of depletion of asubstance in the individual; and (b) determining the blood glucoseconcentration in the individual from the measured depletion rate of thesubstance.
 102. The method according to claim 101 wherein the step ofnon-invasively measuring is performed in the eye.
 103. The methodaccording to claim 102 wherein the step of non-invasively measuring isperformed in the eye by illuminating the eye with light.
 104. The methodaccording to claim 103 wherein the step of non-invasively measuring isperformed in the eye by illuminating the eye with light at a wavelengthabsorbed by the substance being depleted.
 105. The method according toclaim 104 wherein the substance being depleted is all-trans-retinal.106. The method according to claim 104 wherein the substance beingdepleted is rod rhodopsin.
 107. The method according to claim 104wherein the substance being depleted is cone rhodopsin.
 108. A method ofmeasuring the blood glucose concentration in a person, comprising: (a)Providing an apparatus according to claim 89; (b) Deactivating theapparatus after the apparatus has processed optical data to calculatethe blood glucose level a number of times; and (c) Reactivating theapparatus by a health care provider.
 109. The method according to claim108 further comprising: (d) Updating the patient calibration data in thedata storage.
 110. The method according to claim 109 wherein theupdating step is performed by a health care provider using blood samplestaken from the person.
 111. The method according to claim 109 whereinthe updating step is performed periodically.
 112. The method accordingto claim 109 wherein the updating step is performed wirelessly.
 113. Themethod according to claim 109 wherein the updating step is performedusing an access link.
 114. A method for use in the determination of theblood glucose concentration in an individual, comprising: (a) Measuringan indicium of glucose metabolism; (b) Determining the blood glucoseconcentration from the measured indicium.
 115. The method according toclaim 114 wherein the measuring step is performed in the eye.
 116. Themethod according to claim 114 wherein the indicium of the glucosemetabolism is manifested by a change in reflectance.
 117. The methodaccording to claim 114 wherein the indicium of the glucose metabolism isdetected by measuring light reflected from the retina.
 118. The methodaccording to claim 114 wherein the indicium of the glucose metabolism isrepresented by the regeneration of visual pigment.
 119. The methodaccording to claim 114 wherein the indicium of the glucose metabolism isrepresented by the depletion of visual pigment.